Method of screening placental proteins responsible for pathophysiology of preeclampsia, and marker for early diagnosis and prediction of preeclampsia

ABSTRACT

The present invention relates to a method of screening placental proteins responsible for pathophysiology of preeclampsia, and a marker for early diagnosis and prediction of preeclampsia. In accordance with one aspect of the present invention, there is provided a method of screening placental proteins responsible for pathophysiology of preeclampsia by 2D E-proteomics analysis, comprising: isolating placental proteins from a placental tissue; separating the isolated proteins two-dimensionally through 2D electrophoresis; and comparing and analyzing the separated proteins based on scanned gel images and differences in the images between normal placental proteins and preeclamptic placental proteins, wherein the comparison and analysis of the placental proteins based on the scanned gel images and differences in the images are accomplished by selecting proteins with differences of 140% or more between two placentas.

CROSS REFERENCE TO A PARENT APPLICATION

The present application is a divisional application of application Ser.No. 13/154,774 filed on Jun. 7, 2011, which is a divisional applicationof application Ser. No. 12/218,767 filed on Jul. 16, 2008, now U.S. Pat.No. 7,998,508, which claims priority under 35 U.S.C. §119(a) to anapplication filed in the Korean Intellectual Property Office on Jul. 16,2007 and assigned Korean Patent Application No. 10-2007-0071058, thecontents of which are incorporated herein by reference.

SEQUENCE LISTING

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FIELD OF THE INVENTION

The present invention relates to a method of screening placentalproteins responsible for pathophysiology of preeclampsia, and a markerfor early diagnosis and prediction of preeclampsia.

BACKGROUND OF THE INVENTION

Preeclampsia in pregnancy can be a very serious health problem. It cancause fetal growth restriction, fetal death and morbidity, prematuredeliveries, and death of the mother. The exact cause of preeclampsia isnot known, and treatments for efficiently curing or preventingpreeclampsia are not also available yet. Preeclampsia is known to causeseveral problems at the same time, such as high blood pressure(hypertension), pathological edema and leakage of protein into the urine(proteinuria). Further, preeclampsia is one of the pregnancycomplications that bring hypertension, proteinuria and traumatism to themother. It is known that preeclampsia occurs to only about 3-5% ofpregnant women, but it can seriously affect both the mother and herunborn (or newborn) baby, and thus, acts as a major cause of increasingperinatal mortality and morbidity rates.

Globally, at least 200,000 pregnant women die from preeclampsia everyyear. Its symptoms typically become evident after the 20^(th) week ofpregnancy. Preeclampsia is usually diagnosed by detecting high bloodpressure of a pregnant woman or by checking her urine for protein. Earlydiagnosis and timely treatment of preeclampsia can remarkably reducerisks to the mother and her unborn baby, but such a monitoring method byusing those symptoms as criteria is not effective for an early diagnosisof preeclampsia. Further, no treatments are currently available to curepreeclampsia. Preeclampsia can be mild, but potentially life-threateningdepending on the severity of the disease. Despite such clinical risks,however, it is difficult to find the cause or the pathogenesis ofpreeclampsia at an early stage, or to make an early diagnosis andprognosis.

Therefore, if it becomes possible to suggest the pathogenesis ofpreeclampsia and make an early diagnosis and prognosis based on thesame, the mother having preeclampsia and her unborn baby can beprotected, and the death rate would be reduced. Even if many researcheshave been conducted to monitor and predict the occurrence ofpreeclampsia, they are limited to using a specific protein or substance,which is not sufficient to explain the whole phenomenon about theoccurrence of preeclampsia and the pathogenesis thereof.

While the inventors of the present invention have been trying todiscover the pathogenesis of preeclampsia, they checked entire proteinexpressions in a placenta that plays a key role in the onset ofpreeclampsia and analyzed any change in the protein expressions in apreeclamptic placenta. Based on this, they devised a method of screeningplacental proteins responsible for pathophysiology of preeclampsia and amarker for early diagnosis and prediction of preeclampsia and alsosuggested a comprehensive theory of the pathogenesis of preeclampsia tocomplete the present invention.

SUMMARY OF THE INVENTION

It is, therefore, a primary object of the present invention to provide amethod of screening placental proteins responsible for pathophysiologyof preeclampsia.

It is another object of the present invention to provide a markerprotein for early diagnosis and prediction of preeclampsia.

In accordance with one aspect of the present invention, there isprovided a method of screening placental proteins responsible forpathophysiology of preeclampsia by 2D E-proteomics analysis, comprising:isolating placental proteins from a placental tissue; separating theisolated proteins two-dimensionally through 2D electrophoresis; andcomparing and analyzing the separated proteins based on scanned gelimages and differences in the images between normal placental proteinsand preeclamptic placental proteins, wherein the comparison and analysisof the placental proteins based on the scanned gel images anddifferences in the images are accomplished by selecting proteins withdifferences of 140% or more between two placentas.

In accordance with another aspect of the present invention, there isprovided a marker for early diagnosis and prediction of preeclampsia,comprising one or more proteins selected from the protein groupconsisting of chaperonin, ER-60 protease, isocitrate dehydrogenase 1,aldehyde reductase 1, fidaresta chain B bonded to human aldosereductase, voltage-dependent anion channel 1, nuclear chloride channel,cathepsin D chain H, phosphoglycerate mutase 1, endoplasmic reticulumprotein, PSMA2 protein, glutathione S-transferase, Ig heavy chain vregion, smooth muscle myosin alkali light chain, and fatty acid bindingprotein.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the preferred examplesgiven in conjunction with the accompanying drawings, in which:

FIG. 1 shows 2-D gel E-proteomics analysis pictures taken for theidentification of changes in placental proteins of a pregnant woman withpreeclampsia, in which FIG. 1A is a gel picture of placental proteins innormal pregnancy and FIG. 1B is a gel picture of placental proteins inpregnancy with preeclampsia;

FIG. 2 presents comparative pictures of 21 proteins which show bigdifferences in expression between normal placenta and preeclampticplacenta (left-hand side: normal cell; right-hand side: gastric cancercell) in result of the E-proteomics analysis depicted in FIG. 1; and

FIG. 3 offers a conceptual diagram suggesting the pathogenesis ofpreeclampsia on the basis of the analysis of proteins exhibitingdifferent expression in preeclamptic placenta.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following detailed description, reference is made to theaccompanying drawings that show, by way of illustration, specificembodiments in which the invention may be practiced. These embodimentsare described in sufficient detail to enable those skilled in the art topractice the invention. It is to be understood that the variousembodiments of the invention, although different, are not necessarilymutually exclusive. The following detailed description is, therefore,not to be taken in a limiting sense, and the scope of the presentinvention is defined only by the appended claims that should beappropriately interpreted along with the full range of equivalents towhich the claims are entitled.

Then, experiments performed for better understanding the presentinvention will be described in detail as follows, which are set forth toillustrate, but are not to be construed to limit the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention is directed to analyzing protein expression inpreeclamptic placenta. More specifically, the present invention isdirected to analyzing placental proteins that demonstrate more changesin expression in preeclamptic pregnancy than in normal pregnancy, thusidentifying proteins responsible for pathophysiology of preeclampsia.

In order to identify proteins associated with pathophysiology ofpreeclampsia, the inventors conducted 2D E-proteomics analysis on theexpression of placental proteins, and compared protein expression in anormal placenta and in a preeclamptic placenta to verify the differencesin protein expression between them. The “2D E-proteomics analysis”perceives cell or tissue proteins in a packet and identifies an overallchange, not individual changes, in proteins reflected in electrical andphysical natures proved by electrophoresis. This research method is nowactively being used worldwide and is largely composed of primaryseparation of proteins by isoelectric point and secondary gel-basedprotein separation by molecular mass. Images of proteins that arebroadly distributed over the gel through the two-step protein separationtechnique were analyzed by an image analyzer for comparison ofquantitative expression of proteins. Among them, proteins showing bigdifferences were collected, and molecular weight in unit of peptide (asmall cut piece of protein) of which were measured by a molecular weightmeasurement technique called MALDI-TOF MS. Each of the measured peptidemasses was again calculated in terms of the mass of amino acidsconstituting a peptide. The final masses were compared with those in thealready known peptide mass database to prove nature of originalproteins.

The present invention suggests, based on the 2D E-proteomics analysis,21 proteins demonstrating big differences in expression in apreeclamptic placenta. These proteins may be used for explainingabnormal metabolism in a preeclamptic placenta and its pathophysiology.Further, the proteins are expected to be effectively used for earlydiagnosis and prediction of preeclampsia in pregnant women.

For the 2D E-proteomics analysis, the inventors obtained normal placentatissues and preeclamptic placenta tissues and separated placentalproteins according to their isoelectric point (the primary separationstep) and according to their molecular weight (the secondary separationstep). The gel obtained after electrophoresis was stained by silvernitrate. Then, the stained gel was scanned with a flatbed scanner andanalyzed through an image analysis program. Proteins showing differencesof 140% or more in two groups of placental samples were selected andfinally identified. Twenty one proteins were collected and peaks ofprotein mass spectrometry were searched by MASCOT PMF based on the NCBIdatabase (see Table 1). Seventeen out of those twenty one proteins wereanalyzed, and most of them were identified as ones associated withplacental metabolism (see Table 2). These proteins may be categorizedinto different types on the basis of their association with antioxidantactivities, recombination related to stress, apoptosis, glycolysis,immunomodulation, or remodeling of reduced NADP, such that thepathophysiology of protein-underlying preeclampsia can be presented (seeFIG. 3).

Hereinafter, the present invention will be explained in more detailthrough examples. However, it will be apparent to those skilled in theart that these examples are only for the purpose of explaining thepresent invention in detail, but not intended to limit the scope of theinvention.

Example 1 Separation of Placental Proteins

Normal placenta and preeclamptic placenta were prepared for 2DE-proteomics analysis. Placental tissues were ground to fine powder inpresence of liquid nitrogen, and a buffer (tissue: 0.2 g/10 m/) wasadded thereto. The samples were then divided into tubes, boiled for fiveminutes, put into the ice bath for five minutes, and centrifuged at8,000 rpm and 4° C. for 10 minutes. Each of the upper phases wastransferred to a new tube by 800 μl, treated with enzymes (DNase/RNase),and put into the ice bath for 10 minutes. Next, 200 μl of 10%TCA/acetone preparation-50% TCA/acetone was added to each tube. Thetubes were placed into the ice bath for a period of 1 hour. The sampleswere then centrifuged at 12,000 rpm and 4° C. for 10 minutes, and theresulting pallets were washed with acetone. The remaining dry powderswere kept at −20° C.

Example 2 Separation of Proteins by 2D E-Proteomics

<2-1> Primary Separation of Proteins by Isoelectric Point

Proteins are primarily separated based on isoelectric point. Dryimmobilized pH gradient (IPG) strips of 13 cm were added with 250 μlisoelectric point marker containing 50 μg protein and rehydrated over 10hours. The rehydrated IPG strips were subjected to isoelectric pointseparation in an IPG phore (GE Healthcare, USA). The isoelectric pointseparation was carried out for 1 hour at 500 V, for 1 hour at 1,000 V,and finally at 8,000 V until the final accumulated voltage becomes60,000 V. At this time, the highest current was set to 50 μA per strip.The strips separated by isoelectric point were slowly stirred over aperiod of 15 minutes in presence of a primary phase equilibrium solution(50 mM Tris-HCl containing 6M urea, 30% glycerol, 2% SDS, bromophenolblue and 1% DTT, pH 8.8). These primary phase equilibrated strips weresoaked in a secondary phase equilibrium solution (50 mM Tris-HClcontaining 6M urea, 30% glycerol, 2% SDS, bromophenol blue and 2.5%iodoaceamide, pH 8.8) and stirred again over a period of 15 minutes.

<2-2> Secondary Protein Electrophoresis

Proteins are separated in a polyacrylamide gel depending on theirmolecular weight. A 12.5% sodium dodecyl sulfate polyacrylamide gel wasprepared in size of 13 cm through SE 600 Ruby electrophoresis set(Amersham, USA). Phase equilibrated strips were put on the gel, and thegap between the strip and the gel was filled with a sealing Aga. Arunning buffer (25 mM Tris, 192 mM glycine, 2.5 mM SDS, pH 8.3) waspoured into the set, and proteins of the strips were transferred to thegel within the first 20 minutes at 80V, and electrophoresis was carriedout for the next 5 hours at 240V. The electrophoresed gel was stainedwith silver nitrate. In result, by comparing a gel picture of placentalproteins in normal pregnancy by 2D electrophoresis (see FIG. 1A) with agel picture of placental proteins in pregnancy with preeclampsia by 2Delectrophoresis (see FIG. 1B), the inventors could verify changes inplacental proteins in pregnancy with preeclampsia.

Example 3 Gel Scanning and Gel Image Analysis

The stained gel was scanned through a flatbed scanner (UMAX PowerLook1100, USA). During scanning, the option of a transmissive type with 300dpi resolution was chosen. The scanned gel images were analyzed throughan image analysis program (Image Master 2D Platinum, GE Healthcare,USA). Based on the image analysis result, proteins showing differencesup to 140% or more in two groups of placental samples were selected andfinally identified. In result, the inventors checked proteins showingbig differences between normal placenta and preeclamptic placenta byelectrophoresis shown in FIG. 1 (In FIG. 2, left-hand side: normal cell;right-hand side: gastric cancer cell), and identified 21 proteins withdifferences in expression between normal placenta and preeclampticplacenta.

Example 4 Protein Identification

The 21 proteins with differences in expression between normal placentaand preeclamptic placenta used were collected and sent to IN2GEN Co.,Ltd. for protein mass analysis based on MALDI-TOF MS technique. Peaks ofprotein mass spectrometry analyzed were searched by MASCOT PMF based onthe NCBI database and listed with GeneBank IDs in Table 1 below.

TABLE 1 NCBI Spot accession NOs NOs SEQ ID NOs protein identification 149522865 SEQ ID NO: 1 chaperonin 2 1208427 SEQ ID NO: 2 ER-60 protease 31167843 SEQ ID NO: 3 alpha-enolase 4 28178825 SEQ ID NO: 4 Isocitratedehydrogenase 1 5 1633300 SEQ ID NO: 5 Aldehyde reductase 6 493797 SEQID NO: 6 chain B, Fidarestat Bonded to human Aldose reductase 7 14250132SEQ ID NO: 7 Voltage-dependent anion channel 1 8 4588526 SEQ ID NO: 8Nuclear chloride channel 9 5822091 SEQ ID NO: 9 Chain H, Cathepsin D 1056081766 SEQ ID NO: 10 Phosphoglycerate mutase 1 11 5803013 SEQ ID NO:11 Endoplasmic reticulum protein 12 50881968 SEQ ID NO: 12 PSMA2 protein13 2204207 SEQ ID NO: 13 Glutathione S-transferase 14 8249777 SEQ ID NO:14 Ig heavy chain v region 15 16924329 SEQ ID NO: 15 Smooth musclemyosin alkali light chain 16 4557581 SEQ ID NO: 16 Fatty acid bindingprotein

The above Table 1 presents the analysis result on the 21 proteinsobtained by MALDI-TOF MS technique, verifying that all of the 21proteins except one were expressed remarkably high in the preeclampticplacenta compared with the normal placenta.

Example 5 Protein Analysis and Suggestion of Pathophysiology

The inventors analyzed 17 out of the 21 proteins and confirmed that mostof the proteins were associated with placenta metabolism. To be morespecific, the inventors analyzed not only functions of those 21proteins, but also changes in protein expression detected in thepreeclamptic placenta compared with that of the normal placenta, whereinthe analysis result is listed in Table 2 below.

TABLE 2 Relative Category Protein change (%) Structural Smooth musclemyosin alkali light 191 chain Antioxidant and Glutathione S-transferase177 detoxicant Isocitrate dehydrogenase 155 Stress-related Chaperonin(heat shock protein 60) 223 protein remodeling ApoptosisVoltage-dependent anion channel 185 Nuclear chloride channel 208 ChainH, Cathepsin D at pH 7.5 245 Reduced NADP⁺- Aldehyde reductase 142regeneration Chain B, Fidarestat bound to 151 human aldose GlycolysisPhosphoglycerate mutase 267 Alpha enolase 149 Immunoremodeling ER-60protease 179 Other Endoplasmic reticulum protein 29 242 PSMA2 protein156 Ig heavy chain v region 216 Fatty acid binding protein 5 220

The proteins were categorized into different types based on theirassociation with antioxidant activities, recombination related tostress, apoptosis, glycolysis, immunomodulation, or remodeling ofreduced NADP such that the pathophysiology of protein-underlyingpreeclampsia can be presented as shown in FIG. 3.

As discussed above, according to the present invention, the variation incertain protein expression in a preeclamptic placenta can be identifiedby a 2-D E-proteomics analysis, thus making it possible to present atheory on the pathogenesis of preeclampsia. Further, the screeningmethod and marker of the present invention can screen placental proteinsof different expressions in the placenta of a pregnant woman withpreeclampsia, and those proteins can be used as a marker for preventionand early treatment of preeclampsia.

While the invention has been shown and described with respect to thepreferred embodiments, it will be understood by those skilled in the artthat various changes and modifications may be made without departingfrom the spirit and the scope of the invention as defined in thefollowing claims.

1-6. (canceled)
 7. A method of using a marker for early diagnosis and prediction of preeclampsia, comprising: selecting one or more proteins from the protein group consisting of ER-60 protease, aldehyde reductase 1, fidaresta chain B bonded to human aldose reductase, voltage-dependent anion channel 1, nuclear chloride channel, cathepsin D chain H, phosphoglycerate mutase 1, endoplasmic reticulum protein, proteasome subunit alpha type-2 (PSMA2) protein, glutathione S-transferase, Ig heavy chain v region, and smooth muscle myosin alkali light chain, wherein the one or more proteins are isolated from a placental tissue and the isolated proteins are separated two-dimensionally through 2D electrophoresis; and analyzing the isolated and separated proteins based on scanned gel images and differences in the images against normal placental proteins, wherein the marker for early diagnosis and prediction of preeclampsia shows an increased expression of 140% or more than the normal placental proteins based on scanned gel images and differences in the images against the normal placental proteins.
 8. The method of claim 7, wherein the ER-60 protease, aldehyde reductase 1, fidaresta chain B bonded to human aldose reductase, voltage-dependent anion channel 1, nuclear chloride channel, cathepsin D chain H, phosphoglycerate mutase 1, endoplasmic reticulum protein, PSMA2 protein, glutathione S-transferase, Ig heavy chain v region, and smooth muscle myosin alkali light chain have the sequences of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, respectively. 